Measurement and monitoring of cell clonality

Abstract

Methods are provided for the detection and analysis of clonality in a cell population, where parallel sequencing is applied to a nucleic acid sample obtained from the cell population, optionally a population of lymphocytes. Replicate samples are amplified, and sequenced, where identification of coincident sequences in two or more replicates is indicative of clonal expansion.

What is claimed is: 
     
       1. A method of determining the clonal expansion of B cells in response of an individual to a vaccine, the method comprising:
 obtaining a cell sample comprising B cells from said individual in a short defined time period following immunization with said vaccine; 
 dividing said cell sample comprising B cells, or genomic DNA derived therefrom, into at least two distinct pools; 
 amplifying genomic DNA sequences at an immunoglobulin heavy chain locus in said at least two distinct pools with a primer set that amplifies at least 50% of the known rearrangements at the locus; 
 sequencing at least 10 3  reads of the amplified genomic DNA at the locus of interest from said at least two distinct pools; 
 comparing sequences from said amplified genomic DNA to detect the presence of sequences that are coincident in said at least two distinct pools of nucleic acid; 
 wherein the presence of coincident sequences is indicative of clonal expansion and responsiveness to said vaccine. 
 
     
     
       2. A method of determining the clonal expansion of B cells in response of an individual to a vaccine, the method comprising:
 obtaining a cell sample comprising B cells from said individual in a short defined time period following immunization with said vaccine wherein said short defined time period is less than about 14 days; 
 dividing said cell sample comprising B cells, or genomic DNA derived therefrom, into at least two distinct pools; 
 amplifying genomic DNA sequences at an immunoglobulin heavy chain locus in said at least two distinct pools with a primer set that amplifies at least 75% of the known rearrangements at the locus; 
 sequencing at least 10 3  reads of the amplified genomic DNA at the locus of interest from said at least two distinct pools; 
 comparing sequences from said amplified genomic DNA to detect the presence of sequences that are coincident in said at least two distinct pools of nucleic acid; 
 wherein the presence of coincident sequences is indicative of clonal expansion and responsiveness to said vaccine. 
 
     
     
       3. The method of  claim 2  in which the sequences are selected by amplification using oligonucleotide primers set forth in Table 6 with homology to specific regions of the genome. 
     
     
       4. The method of  claim 2 , wherein the short defined time period is less than about 7 days.